Pecam-1 modulation

ABSTRACT

Activation of PECAM-1 (platelet endothelial cell adhesion molecule-1) with, for example, a small molecule or antibody derivative provides a new therapeutic route for modifying platelet activation and thrombus formation which has utility in the treatment or prevention of cardiovascular diseases such as thrombosis, stroke, and vascular occlusion and in the treatment or prevention of homeostasis disorders. This treatment approach activates a general inhibitory mechanism. A screen for activators of PECAM-1 include PECAM-1, an ectodomain of PECAM-1, the cytoplasmic tail of PECAM-1, the ITIM of PECAM-1, an active site of PECAM-1, a recombinant extracellular domain of PECAM-1, or a part of derivative thereof and ways for detecting activation or cross-linking or phosphorylation or tyronsine phosphorylation of PECAM-1, an ectodomain of PECAM-1, the cytoplasmic tail of PECAM-1, the IPIM of PECAM-1, an active site of PECAM-1, a recombinant extracellular domain of PECAM-1, or a part or derivative thereof.

FIELD OF THE INVENTION

The invention relates to the modulation of the activity of plateletendothelial cell adhesion molecule-1 (PECAM-1, CD31) for the treatmentof or for reducing the occurrence of cardiovascular conditions such asthrombosis, vascular occlusion stroke and for the treatment of or forreducing the occurrence of haemostasis disorders.

REVIEW OF THE ART KNOWN TO THE APPLICANT(S)

Thrombosis, an aggregation of blood factors primarily platelets andfibrin with entrapment of cellular elements frequently causing vascularobstruction at the point of its formation, is a remarkably prevalentproblem. It underlies most deaths from cardiovascular disease (whetherthere is another underlying cause or not). The 2001 Heart and StrokeStatistical Update (American Hearth Association) reports the prevalenceof cardiovascular disease to be 1 in 5 Americans with 12.4 millionAmericans currently suffering from coronary heart disease (myocardialinfarction and angina pectoris) and 4.7 million suffer from the effectsof stroke. In 1998 cardiovascular disease (which also includes theeffects of high blood pressure and congestive heart failure) killed915,619 people in America, corresponding to 1 in every 2.5 deaths.

Platelets initiate blood clotting and control the formation of clotsand/or thrombi, rapid and complete activation of platelets at sites oftissue damage is ensured through numerous positive feedback pathways,mainly through the actions of mediators such as thromboxane A₂ and ADPthat are released from activated platelets. The existence of such arapid and reactive system emphasises the need for effective regulationof platelet function in order to prevent disorders such as thrombosisand haemorrhage. Platelet reactivity is a controlled balance betweenpositive and negative regulatory factors and signalling mechanisms.

The use of reagents that target and block activatory platelet cellsurface receptors is known, and has provided a successful approach inthe development of anti-thrombotic drugs. However, due to the complexnature of the homeostasis system and the many ways it can be activated,none of these drugs has produced either a cure or a complete solution toall thrombosis related conditions. At best, in some trials, mortalityhas been reportedly reduced by up to 25%, however numerous side effectsare associated with existing treatments. As a result almost all thesedrugs are used in combination depending on the clinical circumstances,which serves to highlight the need for a safe effective treatment forthrombosis and related cardiovascular conditions.

Much attention has recently been focussed on the identification of thereceptors and signalling pathways that lead to platelet activation,particularly on exposure to collagen, thrombin and ADP.

Despite this intensive research activity the use of an agent whichactivates natural inhibitory receptors so as to modify, or inhibitplatelet activation and thereby reduce thrombus formation has notpreviously been either suggested or attempted.

The inventors have identified a negative regulation system that ismediated via a cell-surface ITIM-bearing adhesion receptor, PECAM-1.PECAM-1 signalling results in a negative feedback on platelet activationpathways and thereby sets the threshold stimulation level for plateletactivation in the absence of injury thus preventing thrombosis..

They have ascertained that activation of PECAM-1 with for example asmall molecule or antibody derivative provides a new therapeutic routefor modifying platelet activation and thrombus formation which hasutility in the treatment or prevention of cardiovascular diseases, suchas thrombosis, stroke and vascular occlusion and in the treatment orprevention of haemostasis disorders.

The present invention provides a far better approach to the treatment ofthese conditions than existing drugs as it activates a generalinhibitory mechanism rather than merely blocking one arm of theactivation system.

In its broadest aspect the invention provides a method of activatingPECAM-1 for modifying or reducing or inhibiting platelet activation, orplatelet aggregation, or platelet secretion.

In a preferred aspect the invention provides a method for activating orcross-linking or phosphorylating PECAM-1 for the treatment of or forreducing the occurrence of cardiovascular diseases such as thrombosis,vascular occlusion or stroke, or for the treatment of or for reducingthe occurrence of haemostasis disorders.

In a further aspect the invention provides an activator for use in thismethod.

In a further aspect the invention provides an activator for thetreatment of or for reducing the occurrence of cardiovascular diseasessuch as thrombosis, vascular occlusion or stroke, or for the treatmentof or for reducing the occurrence of haemostasis disorders.

In another aspect the invention provides an activator for use in themanufacture of a medicament for the treatment of or for reducing theoccurrence of cardiovascular diseases such as thrombosis, vascularocclusion or stroke, or for the treatment of or for reducing theoccurrence of haemostasis disorders.

In another aspect the invention provides a screen for activators ofPECAM-1 comprising PECAM-1, an ectodomain of PECAM-1, the cytoplasmictail of PECAM-1, the ITIM of PECAM-1, an active site of PECAM-1, arecombinant extracellular domain of PECAM-1, or a part or derivativethereof and means for detecting activation or cross-linking orphosphorylation or tyrosine phosphorylation of PECAM-1, an ectodomain ofPECAM-1, the cytoplasmic tail of PECAM-1, the IPIM of PECAM-1, an activesite of PECAM-1, a recombinant extracellular domain of PECAM-1, or apart or derivative thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention will now be described by way of example with reference tothe accompanying experimental data and drawings in which:

FIG. 1. Cross-linking of PECAM-1 inhibits collagen-stimulated plateletaggregation. (A) PECAM-1 cross-linking on platelet surfaces results inits tyrosine phosphorylation and does not stimulate plateletaggregation. (i) PECAM-1 was immunoprecipitated from washed humanplatelets under resting conditions or following PECAM-1 cross-linking.Proteins were separated by SDS-PAGE and immunoblotted to detectphosphotyrosine residues (upper panel). Immunoprecipitation was verifiedby reprobing for PECAM-1 (lower panel). (ii)

PECAM-1 was cross-linked on washed platelets and aggregation wasmonitored using optical aggregometry. (B) (i) Platelets were incubatedwith isotype-matched control IgG for 5 minutes before the addition ofF(ab′)₂ cross-linker for 90 seconds and then were stimulated withcollagen (100 mM (mg/mL)). Aggregation was monitored using opticalaggregometry. (ii) Cross-linking of PECAM-1 inhibits collagen-stimulatedplatelet aggregation. Platelets were stimulated with collagen (100 mM(mg/mL)) for 90 seconds with and without first cross-linking PECAM-1 andplatelet aggregation monitored by optical aggregometry. Data arerepresentative of 3 separate experiments. Tyr(P), tyrosinephosphorylation; PECAM-1 XL, PECAM-1 cross-linking.

FIG. 2. PECAM-1 cross-linking inhibits GPVI- and thrombinreceptor-stimulated platelet aggregation. (A) Platelets were stimulatedwith Cvx at (i) 15 nM (ng/mL) and (ii) 62.5 nM (ng/mL) with and withoutfirst cross-linking PECAM-1, and aggregation responses were monitored byoptical aggregation (arrow indicates the addition of Cvx). (B) Plateletswere stimulated with thrombin at (i) 0.05 U/mL and (ii) 0.1 U/mL withand without prior cross-linking of PECAM-1. Platelet aggregation wasmonitored by optical aggregometry (arrow indicates the addition ofthrombin). Treatment of platelets with isotype-matched control IgGbefore stimulation with Cvx (15 nM (ng/mL)) and thrombin (0.05 U/mL) isshown in A(i) and B(i), respectively. Data are representative of 3separate experiments. PECAM-1 XL, PECAM-1 cross-linking.

FIG. 3. Platelet-dense granule secretion is inhibited by PECAM-1signalling. Platelets were loaded with [³H]5-HT before stimulation withCvx (62.5 nM [ng/mL]) or thrombin (0.1 U/mL). Where required, PECAM-1was cross-linked prior to the addition of an agonist. Secretion of[³H]5-HT into cell medium was measured using scintillation spectrometry.[³H]5-HT release is ex-pressed as a percentage of total tissue contentafter subtraction of basal secretion values. Results represent mean +/−SE (n=3). Student's t-test was used to compare PECAM-1 cross-linked andnon-cross-linked sample for statistical significance. (* indicatesp-values <0.05. PECAM-1 XL, PECAM-1 cross-linking

FIG. 4. PECAM-1 signaling inhibits platelet protein tyrosinephosphorylation. Platelet lysates were prepared in Laemmli buffer fromnonstimulated platelets (stirred with buffer alone) and plateletsstimulated for 90 seconds with Cvx (15 nM [ng/mL]) or thrombin (0.05U/mL). Before stimulation, PECAM-1 was cross-linked in some samples (A)or platelets were incubated with isotype-matched control IgG andcross-linker F(ab′)₂ (B). Proteins were separated by SDS-PAGE underreducing conditions and immunoblotted to detect protein tyrosinephosphorylation. PECAM-1 XL, PECAM-1 cross-linking

FIG. 5. PECAM-1 cross-linking inhibits the mobilization of calcium fromintracellular stores. Fura-2 AM loaded human platelets were stimulatedwith either Cvx or thrombin, and the mobilization of calcium wasmeasured fluorometrically (arrow indicates the addition of agonist).Panel A) Treatment of platelets with control IgG and cross-linkerF(ab′)₂ has no effect on Cvx- and thrombin-stimulated calciummobilization.

FIG. 6: Panels B i and ii) show representative calcium responses forstimulation with Cvx at 62.5 nM (ng/mL) and 15 nM (ng/mL), respectively,with and without first cross-linking PECAM-1. Traces are representativeof 3 separate experiments. Panel (iii) shows PECAM-1-induced percentagereduction in peak levels of intracellular calcium stimulated by 62.5,31.25, and 15 nM (ng/mL) Cvx. Data presented represents mean +/− SE(n=3).

FIG. 7: Panels C i, and ii) show representative calcium responses forstimulation with thrombin at 0.1 U/mL and 0.05 U/mL, respectively, withand without first cross-linking PECAM-1. Traces are representative of 3separate experiments. Panel (iii) PECAM-1-induced percentage reductionin peak levels of intracellular calcium stimulated by 0.5, 0.1, and 0.05U/mL thrombin. Data represent mean +/− SE (n=3)). PECAM-1 XL, PECAM-1cross-linking.

Platelet endothelial cell adhesion moleule-1 PECAM-1 is a 130 kDamembrane-spanning glycoprotein whose expression is restricted to severalhaematopoietic cell types including platelets, monocytes, neutrophils,certain T-lymphocytes and also vascular endothelial cells. The functionsof PECAM-1 are diverse and include angiogenesis, vasculogenesis,integrin regulation, transendothelial migration of leukocytes, and T-and B-cell antigen receptor function, although the role of this moleculein platelets is presently unclear. When PECAM-1 was cloned it wasassigned to the family of cell adhesion molecules on the basis ofstructural similarities. PECAM-1 is involved in adhesion although muchattention has been recently been directed to studying its ability toparticipate in signal transduction. The cytoplasmic tail of PECAM-1contains a conserved motif called an immunoreceptor tyrosine basedinhibitory motif (ITIM), which underlies its signaling properties and isshared by a growing family of inhibitory receptors. These include theimmunoglobulin G receptor FcγRIIB, the killer inhibitory receptors (KIR)and signal regulatory proteins (SIRPs), although PECAM-1 is the onlyITIM-bearing receptor that has been reported to be expressed inplatelets. It has therefore been proposed that PECAM-1 should beassigned to the Ig-ITIM family of receptors.

The ligand binding properties of PECAM-1 are complex. It has thecapacity for homophilic interactions, and also heterophilic interactionswith a number of molecules that include integrin α_(v)β₃ and CD38,PECAM-1 becomes phosphorylated on tyrosine residues in response to avariety of stimuli that include PECAM-1 cross-linking, activation of thehigh affinity receptor for immunoglobulin E (FcεRI), shear and oxidativestress. The inventors have recently found that platelet activation viathe collagen receptor glyoprotein GPVI (GPVI) and thrombin receptorsresult in PECAM-1 tyrosine phosphorylation which is not dependent onplatelet aggregation and secretion, although tyrosine phosphorylation isenhanced by aggregation. The tyrosine residues that becomephosphorylated in PECAM-1 have been mapped and fall within the ITIM.Phosphorylated ITIMs recruit signalling molecules such as the tyrosinephosphatases SHP-1 and SHP-2 that bind to the motif via Src-homology 2domain interactions. Indeed, both SHP-1 and SHP-2 have been shown toassociate with tyrosine phosphorylated PECAM-1, and PECAM-1 ITIMphosphopeptides activate these phosphatases in vitro. Generally, theseprotein tyrosine phosphatases exhibit inhibitory effects bycounteracting tyrosine kinase-dependent pathways, although SHP-2 hasbeen shown to positively regulate growth factor receptor signalling.

Immunoreceptor tyrosine-based activatory motif-(ITAM) bearing receptorshave been shown to have a critical place in the regulation of plateletfunction. Indeed the collagen receptor GPVI-FcR γ-chain complex signalsthrough an ITAM present on the cytoplasmic tail of the FcR γ-chain.Several studies in other cell systems have provided evidence of anantagonistic relationship between ITAM and ITIM containing receptorswhen expressed on the same cell. An example of this is the receptor forIgG FcγRIIB (ITIM) that negatively regulates cell activation stimulatedby FcγRIIA (ITAM).

PECAM-1 was stimulated through cross-linking using antibodies directedto the extracellular domain of the receptor. This strategy was thechosen as the most specific manner to activate PECAM-1, and activationwas confirmed since cross-linking stimulated its tyrosinephosphorylation. PECAM-1 cross-linking stimulates tyrosinephosphorylation (FIG. 1) and association of SHP-2 (not shown) but doesnot itself cause platelet activation. Tyrosine phosphorylation ofPECAM-1 on cross-linking was shown not to be dependent on integrinα_(iib)β₃ engagement. Cross-linking PECAM-1 for 90 s prior tostimulation with collagen caused inhibition of platelet aggregation. Atlower concentrations of collagen, aggregation was inhibited completely,but even at very high concentrations of collagen (100 μg/ml) PECAM-1activation caused a substantial inhibition of aggregation. Sincecollagen is also able to bind other receptors on the platelet, includingthe integrin α₂β₁, the effect of PECAM-1 cross-linking on GPVI-mediatedplatelet aggregation using the specific agonist Cvx was examined.Similar results were obtained, with complete inhibition of aggregationat lower concentrations of agonist (15 ng/ml) and partial effects athigher concentrations (62.5 ng/ml), indicating that PECAM-1 is a potentinhibitor of GPVI-mediated (ITAM) platelet activation.

Experiments were coducted to determine whether the inhibitory effect ofPECAM-1 is restricted to signalling via ITAM containing receptors. Onthe contrary, PECAM-1 cross-linking was also found to inhibitthrombin-stimulated platelet aggregation. Although no inhibitory effectof PECAM-1 was observed at moderate concentrations of thrombin (0.5 and1 U/ml, results now shown) dramatic levels of inhibition were observedat very high concentrations of collagen (100 μg/ml). PECAM-1 mediatedinhibition of thrombin-stimulated aggregation was observed only a lowerthrombin concentrations (complete inhibition with 0.05 U/ml and slightinhibition at 0.1 U/ml). Showing that PECAM-1 activation inhibitsthrombin-stimulated platelet aggregation less efficiently thancollagen-stimulated aggregation.

The effect of PECAM-1 cross-linking on platelet aggregation stimulatedby other G protein-coupled receptor agonists was also examined.Aggregation in response to low concentrations of the thromboxane mimeticU46619 were also reduced by PECAM-1 signalling. In addition, preliminarywork suggests that ADP-induced platelet aggregation at low agonistconcentrations may also be affected.

The inventors have demonstrated that activation of PECAM-1 signalling byantibody-mediated cross-linking results in inhibition ofcollagen-mediated activation. They have also shown that the effects ofthe GPVI-selective agonist convulxin, are inhibited by activation ofPECAM-1 signalling. Thrombin-stimulated activation has also been shownto be inhibited by the activation of PECAM-1 signalling, indicating thatthe inhibitory effects of PECAM-1 are not restricted to the inhibitionof ITAM-containing receptor signalling pathways.

This is consistent with PECAM-1 performing a negative regulatory role inthe control of platelet activation stimulated by both ITAM- andnon-ITAM-containing receptor agonists.

Even though previous studies by for example Duncan G S, Andrew D P,Takimoto H, et al: Genetic evidence for functional redundancy ofplatelet/endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficientmice reveal PECAM-1-dependent and PECAM-1-independent functions. J.Immun. 1999; 162:3022-3030, and Patil S, Newman D K, Newman P J: PECAM-1serves as an inhibitory receptor that modulates platelet responses tocollagen. Blood. 2001; 97:1727-1732, have reported that thrombin and ADPstimulated platelet aggregation in PECAM-1 -deficient mouse platelets isnormal.

The observation that ITAM-mediated signalling is inhibited to a higherdegree than non-ITAM-mediated signalling is further supported byexperiments in platelets where PECAM-1 was co-ligated with FcγRIIA (anITAM-containing immunoglobulin G receptor). Co-ligation results ininhibition of FcγRIIA-mediated platelet aggregation and intracellularcalcium mobilisation stimulated by receptor cross-linking.

The inhibitory effects of PECAM-1 on stimulation with agonists such asthe collagen, Cvx, thrombin, and the thromboxane mimetic U46619, suggestthat PECAM-1 inhibits primary signalling events and also secondarystimulation by factors released by activated platelets. It is possible,therefore, that inhibition of receptors that cross-talk withGPVI-mediated signalling may contribute to the inhibitory actions ofPECAM-1 on collagen- and convulxin-stimulated activation. This may alsoexplain why the inhibitory functions of PECAM-1 on GPVI-mediatedactivation are more effective than on thrombin-mediated activation.

Having established that PECAM-1 cross-linking inhibits plateletfunction, the effect of this on some aspects of signal transduction wasexamined. Platelet activation was found to be accompanied by aconcomitant inhibition of platelet protein tyrosine phosphorylation anddecreased levels of calcium mobilisation from intracellular stores.Platelet activation by the collagen receptor GPVI is dependent ontyrosine kinases, and consequently is associated with the rapid tyrosinephosphorylation of a wide variety of platelet proteins. Stimulation withthrombin results in protein tyrosine phosphorylation, but to a lesserdegree. Cross-linking PECAM-1, which in itself does not alter proteintyrosine phosphorylation levels, inhibits substantially the level oftyrosine phosphorylation that is induced by subsequent stimulation withCvx or thrombin. This is consistent with the reduction in aggregationand secretion observed. The identities of the phosphoproteins whosephosphorylation is reduced on PECAM-1 cross-linking is currently underinvestigation.

Underlying the PECAM-1 mediated inhibition of platelet activation, is asignificant level of inhibition of calcium release from intracellularstores. As seen with aggregation assays, at lower concentrations of Cvxand thrombin (15 ng/ml) ad 0.05 U/ml, respectively) PECAM-1 signallinginhibits release almost completely, where a partial effect is observedat higher agonist concentrations. Calcium mobilisation is stimulatedthrough the intracellular generation of inositol 1,4,5-trisphosphate(IP₃) from phosphatidylinositol 4,5-bisphosphate by phospholipase C. Itis well established that in platelets, stimulation with collagen leadsto phosphorylation and activation of PLCγ2 isoform, and thrombinsignalling regulates PLC_(β). PECAM-1 cross-linking results in asubstantial and significant reduction in total inositol phosphatesstimulated by Cvx and thrombin. Which suggests that PECAM-1 exerts itseffects on collagen- and thrombin-stimulated signalling either upstreamof the PLC isoforms, or on these enzymes themselves.

Methods

Materials—Horm-Chemie collagen (collagen-fibres from equine tendons) waspurchased from Nycomed (Munich, Germany). Convulxin was purified fromthe venom of the rattlesnake (Crotalus durissus terrificus) as describedpreviously. The thromboxane mimetic U46619 and ADP was purchased fromSigma (Poole, UK). Anti-phosphotyrosine monoclonal antibody (4G10) wasfrom Upstate Biotechnology (TCS Biologicals, Buckinghamshire, UK).Anti-PECAM-1 antibodies: monoclonal antibody HCl/6 was from Serotec(Oxford, UK); polyclonal anti-PECAM-1 (C-20) and monoclonal antibodyAB468 were from Autogen Bioclear Ltd. (Wiltshire, UK); monoclonalantibody PECAM 1.3 was kindly provided by Professor Peter Newman (TheBlood Centre of Woutheastem Wisconsin, Milwaukee, Wis., USA). Controlmouse IgG, was purchased from Sigma (Poole, UK). Monoclonal antibodyIV.3 was purified from hybridoma cell culture medium and F(ab′)₂fragments generated by pepsin digestion using reagents purchased fromPierce (Perbio Scientific, Chester, UK). Horseradishperoxidase-conjugated secondary antibodies and the enhancedchemiluminescence detection system were purchased from AmershamPharmacia Biotech (Buckinghamshire, UK). Fura-2 AM was from MolecularProbes (Cambridge Bioscience, Cambridge, UK).

Preparation and stimulation of Platelets: Human platelets from drug-freevolunteers were prepared on the day of the experiment by differentialcentrifugation and suspended in modified Tyrodes-Hepes buffer (134 mMNaCl, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 20 mM Hepes, 5 mMglucose, 1 mM MgCl₂, pH7.3) to a density of 2×10⁸ cells/ml. For proteinprecipitation experiments, platelets were resuspended at 8×10⁸ cells/mlin buffer containing 1 mM EGTA to prevent aggregation. Stimulation ofplatelets (450 μl) with coliagen, convulxin (Cvx) and thrombin(delivered in 50 μl) was performed at 37° C. in an optical plateletaggregometer (Chrono-log Corporation) with continuous stirring (1200rpm). PECAM-1 activation was stimulated by incubation with anti-PECAM-1antibodies (AB468, 1 μ/ml or PECAM 1.3, 10 μg/ml) for 5 minutes,followed by incubation for 90 s with F(ab′)₂ fragments of anti-IgGsecondary antibodies (30 μg/ml) to cross-link. mAb AB468 was generatedagainst full length PECAM-1 ectodomain. Control experiments wereperformed using an irrelevant isotype-matched antibody in place of AB468or PECAM-1.3 and used at the same concentration. In some experiments thelow affinity receptor for IgG was blocked by incubation with asaturating concentration (1 μg/ml) of F(ab′)₂ fragments of mAbIV.3 for 5minutes. Saturating concentrations of mAbIV.3 antibody were establishedby determining the concentration of F(ab′)₂ fragment that completelyinhibited subsequent whole IgG-mediated FcγRIIA cross-linking andplatelet activation. Platelet aggregation was determined by opticalaggregometry.

Immunoprecipitation studies: Platelet stimulation was terminated by theaddition of an equal volume of ice cold lysis buffer (2% [v/v] NonidetP40, 20 mM Tris, 300 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 2 mM Na₃VO₄, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/mlpepstatin A, pH7.3). Detergent-insoluble debris was removed and thelysates were pre-cleared by mnixing with protein A-Sepharose for 1 hourat 4° C. (20 μl of a 50% [w/v] suspension of protein A-Sepharose ofTris-buffered saline-Tween [TBS-T: 20 mM Tris, 137 mM NaCl, 0.1% [v/v]Tween 20, pH7.6). Protein A-Sepharose was removed from the lysatesbefore the addition of anti-PECAM-1 antibody (HCl/6, 1 μg). Followingrotation at 4° C. for 1 hour, 0.5 μl secondary antiserum was added(rabbit anti-mouse IgG) and mixed for a further 30 min. 25 μl proteinA-Sepharose suspension was added to each sample and mixing continued for1 hour before washing the Sepharose pellet in lysis buffer followed by awash with TBS-T, and the addition of Laemmli sample-treatment buffer.Proteins were separated by SDS-PAGE under reducing conditions using 10%gels and transferred to polyvinylidene difluoride membranes by semi-drywestern blotting.

Immunoblotting: Membranes were blocked by incubation in 10% (w/v) bovineserum albumin (BSA) dissolved din TBS-T. Primary and secondaryantibodies were diluted in TBS-T containing 2% (w/v) BSA, and incubatedwith membranes for 1 hour at room temperature. Blots were washed for 2hours in TBS-T following each incubation with antibodies for 1 hour atroom temperature, and then developed using an enhanced chemiluminescencedetection system. Primary antibodies were used at a concentration of 1μg/ml (anti-phosphotyrosine, 4G10; anti-PECAM-1, C-20) and horseradishperoxidase-conjugated secondary antibodies were diluted 1:10000.

5-Hydroxytryptamine (5-HT) secretion assay: Platelets were loaded with[³H]5-HT by incubation with 0.5 μCi/ml of platelet-rich plasma for 1 hat 37° C. Platelets were prepared from the platelet-rich plasma asdescribed above. Stimulation of platelets was terminated by addition ofan equal volume of 6% glutaraldehyde and microcentrifugation, and thelevel of [³H]5-HT release into the supernatant was determined byscintillation spectrometry. [³H]5-HT release was expressed as apercentage of the total tissue content following subtraction of releaseunder basal conditions.

Measurement of [Ca²⁺]₁ by spectrofluorimetry: Washed human platelets(prepared as above) were incubated at 2×10⁹ cells/ml in calcium freeTyrodes-hepes buffer with 3 μM Fura-2 AM for 45 min. Platelets werewashed once and resuspended at 2×10⁸ cells/ml in modified Tyrodes-hepesbuffer. Stimulation of platelets (450 μl) in the presence of 2 mM EGTAwith Cvx and thrombin (delivered in 50 μl) was performed with constantstirring at 37° C. in a luminescence spectrophotometer (LS-50B, PerkinElmer) with excitation wavelengths of 340 nm and 380 nm. Fluorescenceemission was measured at a wavelength of 510 nm. Where required, PECAM-1was cross-linked prior to stimulation with agonist as described above.The ration of emission values (excitation at 340/380 nm) was calculatedand converted to calcium concentration using FLWinLab software (PerkinElmer) utilising the equation [Ca²⁺]₁ =K_(d)×(R−R_(min))/R_(max)−R)×SFB(where R is emission ratio value (340/380 nm). R_(max) the maximum340/380 ratio, was determined by lysing platelets with 25 μM digitoninin the presence of 1 mM CaCl₂. The R_(min) 340/380 ratio was obtained byaddition of 2 mM EGTA. K_(d) is the dissociation constant of theFura-2/Ca²⁺ complex (224 nM) and SFB is the fluorescence ratio at340/380 nm of R_(min) and R_(max).

Statistical Analysis: Determination of statistical significance wasperformed using Student's paired T-test. The results are expressed asthe mean +/− standard error of the mean (S.E.M.).

Experimental Data

Cross-linking PECAM-1 inhibits collagen-stimulated platelet aggregation:An antagonistic relationship has been reported between ITIM- andITAM-containing receptors when expressed in the same cell. Since theplatelet collagen receptor GPV1 signals through an ITAM present on theFcR γ-chain with which it is associated, the inventors investigated theeffect of PECAM-1 signalling on platelet activation with collagen.PECAM-1 was activated by incubation with antibodies specific for theectodomain of PECAM-1 (AB468 [FIG. 1] or PECAM-1.3 [not shown]) andcross-linked with a secondary antibody [F(ab′)₂ fragment]. This resultedin increased tyrosine phosphorylation of the protein, and did not resultin stimulation of platelet aggregation (FIG. 1A). Tyrosinephosphorylation of PECAM-1 was maintained on cross-linking in thepresence of EGTA (1 mM), RGDS peptide (0.5 mM) and the γ-chain peptideof fibrinogen (100 μM) added separately or altogether (not shown). This,together with the fact that these experiments were performed on washedplatelets indicates that the tyrosine phosphorylation of PECAM-1 oncross-linking is not dependent on integrin α_(IIb)β₃ engagement. Theeffect of PECAM-1 cross-linking for 90 s prior to stimulation withcollagen was found to have a marked inhibitory effect oncollagen-stimulated platelet aggregation. At lower concentrations ofcollagen (e.g. 10 μg/ml), cross-linking of PECAM-1 before agonistaddition completely abolished aggregation (data not shown). FIG. 1B(ii)shows the marked inhibitory effect of PECAM-1 cross-linking on a veryhigh concentration of collagen (19 μg/ml). The use of an isotype-matchedIgG control and cross-linker F(ab′)₂, had no effect of PECAM-1 tyrosinephosphorylation (not shown) and collagen-stimulated platelet aggregation[FIG. 1B(i)]. Results are representative of 3 separate experiments.Similar results were obtained using the alternative anti-PECAM-1antibody PECAM 1.3. In some experiments the low affinity receptor forIgG FcγRIIA was blocked prior to PECAM-1 cross-linking and agoniststimulation using a saturating concentration of F(ab′)₂ fragments ofmAbIV.3. The inhibitory effect of PECAM-1 cross-linking was unalteredunder these conditions. This clearly indicates that the inhibitoryeffect of PECAM-1 using antibodies is not due to activation of FcγRIIA.

PECAM-1 cross-linking inhibits GPVI- and thrombin receptor-mediatedplatelet aggregation: Given the marked effect of PECAM-1 signalling oncollagen-mediated platelet aggregation the inventors investigatedwhether this effect was restricted to GPVI-mediated signalling only.GPVI was stimulated using the selective agonist Cvx, a protein purifiedfrom the venom of the rattlesnake Crotalis durissus terrificus.Aggregation stimulated with 15 ng/ml Cvx was completely inhibited byprior activation of PECAM-1 [FIG. 2A(i)], and a partial inhibitoryeffect was observed at higher concentrations of Cvx [31.25 and 62.5ng/ml, not shown and in FIG. 2A(ii), respectively]. Similar results wereobserved on stimulation of platelets with the G protein coupled receptoragonist thrombin. Complete inhibition of aggregation at 90 s stimulationwas observed at a thrombin concentration of 0.05 U/ml [FIG. 2B(i) and apartial effect at 0.1 U/ml FIG. 2B(ii)]. The use of an isotype-matchedIgG control and cross-linker F(ab′)₂ had no effect on Cvx- orthrombin-stimulated platelet aggregation [FIG. 2A(i) and 2B(i)]. Noinhibitory effect of PECAM-1 activation was observed at higherconcentrations of thrombin (e.g. 0.5 and 1 U/ml, data not shown).Results are representative of 5 separate experiments. Similar resultswere obtained using the alternative anti-PECAM-1 antibody PECAM-1.3, andwhen FcγRIIA was blocked prior to PECAM-1 cross-linking.

Platelet secretion is inhibited by PECAM-1 signalling: Plateletactivation is accompanied by secretion from dense-granules. Densegranule secretion was assessed by measuring the release of [³H]5-HT frompre-loaded washed platelets. FIG. 3 shows the results of experiments todetermine the effect of PECAM-1 cross-linking on [³H]5-HT secretion. Asignificant reduction in secretion was observed in platelets wherePECAM-1 was activated before stimulation with Cvx (81.9% +/−2.9 to 39.8%+/−4.1, p=0.02, n=3) or thrombin (70.9% +/−4.6 to 37.0% +/−8.0, p=0.01,n=3). The use of an isotype-matched IgG control and cross-linker F(ab′)₂had no effect on Cvx- or thombin-stimulated dense granule secretion (notshown). Experiments performed in the presence of mAb IV.3 to block theFc receptor FcγRIIA produced similar results.

PECAM-1 inhibits platelet protein tyrosine phosphorylation: The effectof PECAM-1 cross-linking on GPVI- and thrombin receptor-stimulatedsignalling was also investigated. Platelets were stimulated with Cvx (15ng/ml) or thrombin (0.05 U/ml) with or without prior cross-linking ofPECAM-1 for 90 s. Whole cell protein tyrosine phosphorylation levelswere determined by immunoblotting. Cross-linking PECAM-1 alone had noeffect on basal platelet tyrosine phosphorylation levels (FIG. 4).Stimulation with Cvx (15 ng/ml) or thrombin (0.05 U/ml) caused anincrease in the level of tyrosine phosphorylation of a broad range ofproteins. In those samples where PECAM-1 signalling was stimulated bycross-linking before incubation with Cvx or thrombin, total tyrosinephosphorylation was reduced (FIG. 4). The use of an isotype-matched IgGcontrol and cross-linker F(ab′)₂ had no detectable effect on Cvx- orthrombin-stimulated total tyrosine phosphorylation levels.

PECAM-1 inhibits the mobilisation of calcium from intracellular stores:Stimulation of the collagen receptor GPVI and thrombin receptors leadsto rapid intracellular mobilisation of calcium, an effect that isessential for secretion and aggregation. Intracellular calcium levelswere measured fluorometrically using the calcium sensitive dye Fura-2AM. Experiments were performed in the presence of 2 mM EGTA to preventthe entry of extracellular calcium. Stimulation of platelets with Cvxand thrombin resulted in a rapid increase in the levels of intracellularcalcium that declined over a period of approximately 5 minutes. Theincubation of platelets with control antibody ad cross-linker F(ab′)₂caused no change in Cvx- and thrombin-stimulated intracellular calciummobilisation (FIG. 5). The cross-linking of PECAM-1 for 90 s beforeincubation with Cvx (62.5 ng.ml) or thrombin (0.1 U/ml) resulted in amarkedly reduced level of calcium mobilisation [FIGS. 6B(i) and 7C(i)].At the lower concentrations of agonists used (Cvx, 15 ng/ml; thrombin,0.05 U/ml) calcium mobilisation was almost abolished [FIGS. 6B(ii) and7C(ii)]. The effect of PECAM-1 cross-linking on the reduction of peakintracellular calcium levels for a range of agonist concentrations isshown in FIGS. 6B(iii) and 7C(iii). A reduction of at least 50% incalcium mobilisation was observed at all of the concentrations of Cvxand thrombin tested. Similar results were obtained using the alternativeanti-PECAM-1 antibody PECAM-1.3. Furthermore, experiments performed inthe absence of extracellular EGTA indicate that PECAM-1 cross-linkingdoes not inhibit agonist-induced influx of calcium (data not shown).

1-18. (canceled)
 19. The activation of PECAM-1 for modifying or reducingor inhibiting platelet activation, or platelet aggregation, or plateletsegregation.
 20. The activation claimed in claim 19 wherein theactivation comprises cross-linking PECAM-1.
 21. The activation claimedin claim 19 wherein the activation comprises antibody mediatedcross-linking.
 22. The activation claimed in claim 21 wherein theantibody is specific for the ectodomain of PECAM-1.
 23. The activationclaimed in claim 21 further comprising a secondary antibody.
 24. Theactivation claimed in claim 19 wherein the activation comprisesphosphorylation of PECAM-1.
 25. The activation claimed in claim 24wherein the phosphorylation occurs at the cytoplasmic tail of PECAM-1.26. The activation claimed in claim 24 wherein the phosphorylationoccurs within the ITIM of PECAM-1.
 27. The activation claimed in claim24 wherein PECAM-1 is phosphorylated at tyrosine residues.
 28. Theactivation of cross-linking or phosphorylation of PECAM-1 claimed inclaim 19 for the treatment of or for reducing the occurrence ofcardiovascular diseases such as thrombosis, vascular occlusion orstroke, or for the treatment of or for reducing the occurrence ofhaemostasis disorders.
 29. The activation claimed in claim 19 whereinthe activation or cross-linking or phosphorylation of PECAM-1 modifiesor inhibits or decreases any one selected from the group comprising:total tyrosine phosphorylation, platelet protein phosphorylation,platelet secretion from dense granules, mobilization of calcium fromintracellular stores, production of inositol phosphates, and regulationof integrin-linked kinase.
 30. The activation or cross-linking orphosphorylation of PECAM-1 as claimed in claim 19 for inhibiting ormodifying or reducing platelet activation stimulated by ITAM or non-ITAMcontaining receptors or receptor agonists.
 31. The activation orcross-linking or phosphorylation of PECAM-1 as claimed in claim 30 forinhibiting or reducing or modifying the activation, aggregation orsecretion of platelets in response to any one selected from the groupcomprising; collagen, collagen related peptide (CRP), convuluxin,thrombin, ADP, thromboxane mimetics, U46619, immunoglobulin G FcγRIIA(FcγRIIA), immunoglobulin E FcεRI (FcεRI), tyrosine kinase,GPVI-mediated signalling and thrombin receptor mediated signalling. 32.A PECAM-1 activator for use in accordance with claim
 19. 33. Anactivator as claimed in claim 32 wherein the activator is selected fromthe group comprising; a small molecule, an antibody, an antibodyderivative, an agonist, an antagonist, a ligand, a DNA sequence, acomplementary DNA sequence, an antisense DNA sequence, a probe, aprotein sequence, a recombinant extracellular domain or domains ofPECAM-1, a catalyst, shear, oxidative stress, FcεRI, the high affinityreceptor for FcεRI, an activated form of the high affinity receptorFcγRIIA, FcγRIIA, the low affinity receptor for FcγRIIA and an activatedform of the low affinity receptor FcγRIIA.
 34. The activator claimed inclaim 32 for the treatment of or for reducing the occurrence ofcardiovascular diseases such as thrombosis, vascular occlusion orstroke, or for the treatment of or for reducing the occurrence ofhaemostasis disorders.
 35. The activator claimed in claim 32 for the usein the manufacture of a medicament for the treatment of or for reducingthe occurrence of cardiovascular diseases such as thrombosis, vascularocclusion or stroke, or for the treatment of or for reducing theoccurrence of haemostasis disorders.
 36. A screen for activators ofPECAM-1 comprising PECAM-1, an ectodomain of PECAM-1, the cytoplasmictail of PECAM-1, the ITIM of PECAM-1, an active site ofPECAM-1, arecombinant extracellular domain or domains of PECAM-1, or a part orderivative thereof and means for detecting activation or cross-linkingor phosphorylation or tyrosine phosphorylation of PECAM-1, an ectodomainof PECAM-1, the cytoplasmic tail of PECAM-1, the IPIM of PECAM-1, anactive site of PECAM-1, a recombinant extracellular domain or domains ofPECAM-1 or a part or derivative thereof.